Abstract

Cynomolgus monkeys are a commonly used species in preclinical drug discovery, and have high genetic similarity to humans, especially for the drug-metabolizing cytochrome P450s. However, species differences are frequently observed in the metabolism of drugs between cynomolgus monkeys and humans, and delineating these differences requires expressed CYPs. Toward this end, cynomolgus monkey CYP3A4 (c3A4) was cloned and expressed in a novel human embryonic kidney 293-6E cell suspension system. Following the preparation of microsomes, the kinetic profiles of five known human CYP3A4 (h3A4) substrates (midazolam, testosterone, terfenadine, nifedipine, and triazolam) were determined. All five substrates were found to be good substrates of c3A4, although some differences were observed in the Km values. Overall, the data suggest a strong substrate similarity between c3A4 and h3A4. Additionally, c3A4 exhibited no activity against non-h3A4 probe substrates, except for a known human CYP2D6 substrate (bufuralol), which suggests potential metabolism of human cytochrome CYP2D6-substrates by c3A4. Ketoconazole and troleandomycin showed similar inhibitory potencies toward c3A4 and h3A4, whereas non-h3A4 inhibitors did not inhibit c3A4 activity. The availability of a c3A4 preparation, in conjunction with commercially available monkey liver microsomes, will support further characterization of the cynomolgus monkey as a model to assess CYP3A-dependent clearance and drug-drug interactions.

Introduction

During drug discovery and development, heavy reliance is placed on utilizing preclinical species to predict human pharmacokinetics (PK), pharmacology, and toxicity. Cynomolgus monkeys (Macaca fascicularis) are extensively used in this regard since they are one of the phylogenetically closest species to humans, apart from nonhuman primates. A comprehensive assessment has suggested that human PK is most reliably predicted from monkeys more so than from rats or dogs, which show a significantly different drug-metabolizing enzyme homology from humans (Ward and Smith, 2004). Among drug-metabolizing enzymes, cytochrome P450s (P450s) play the most significant role in determining human PK parameters, and hence understanding monkey-human P450 species differences is critical to predicting human exposures of novel chemical entities based on monkey data. Alternatively, excluding monkey data from a human PK prediction can be accomplished in a logical, mechanistic manner if monkey-human species differences are proven for the implicated P450. Given that greater than 90% homology exists for most of the P450s between the two species, cynomolgus monkey CYP2C20, 2C43, 2C75, and 3A8 have been renamed CYP2C8, 2C9, 2C19, and 3A4, respectively (Iwasaki and Uno, 2009; Emoto et al., 2013). In addition, it has been proposed that 2B17 and 2B30 be renamed to 2B6, 2C74 to 2C8, 2C83 to 2C9, 2F6 to 2F1, 3A64 to 3A4, 3A66 to 3A5, and 4F45 to 4F2 (Uno et al., 2011).

While excellent homology between human and cynomolgus monkey P450s would indicate similar PK of their respective substrates, a number of studies have found that cynomolgus monkeys are characterized by higher first-pass metabolism than humans (Sietsema, 1989; Chiou and Buehler, 2002; Ward and Smith, 2004; Nishimura et al., 2007; Komura and Iwaki, 2008; Takahashi et al., 2009, 2010). One study found that 75% of 16 tested compounds in cynomolgus monkeys showed significantly lower bioavailability than humans, even though clinically these are orally administered drugs (Takahashi et al., 2009; Nishimuta et al., 2011). The authors showed that the fraction of oral dose absorbed was similar between species, whereas the fraction escaping intestinal (gut) metabolism (Fg) in cynomolgus monkeys was close to zero, which was corroborated by higher intrinsic clearance in cynomolgus monkey intestinal microsomes as compared with human intestinal microsomes. Additionally, a much higher intestinal extraction was observed in monkeys, as compared with humans, for midazolam (MDZ), nifedipine, amitriptyline, propranolol, and timolol, drugs metabolized by human CYP3A4 (h3A4), 2C19, 2D6, and 1A2, respectively, suggesting that the lower Fg in cynomolgus monkeys is not exclusive to h3A4 substrates (Akabane et al., 2010; Yoda et al., 2012). Because of the high levels of CYP3A5 expression in the jejunum, it has been speculated that CYP3A5 could play an important role in the low cynomolgus monkey Fg (Nishimuta et al., 2011). Additionally, total P450 content (based on the carbon monoxide difference spectrum) is ∼700 pmol/mg in cynomolgus monkeys, but the immunoquantitated amount of CYP3A4 therein is lower (∼100 pmol/mg). In contrast, the ratio of spectral to immune-quantified levels of CYP3A4 in human liver microsomes (HLM) is less than 2 (Uehara et al., 2010; Emoto et al., 2013). This raises the possibility that novel cynomolgus monkey CYPs such as CYP4A and CYP4F may be abundantly expressed in cynomolgus monkeys, which may contribute further to species differences (Uehara et al., 2010; Emoto et al., 2013).

Approximately 75% of the drugs on the market are cleared by P450s, and CYP3A subfamily members are responsible for the metabolism of more than 50% of such drugs. The homology between cynomolgus CYP3A4 (c3A4) and h3A4 is 93% (amino acid sequence), while for cynomolgus CYP3A5 (c3A5) and human CYP3A5, the homology is 91% (amino acid) (Emoto et al., 2013). The excellent amino acid homology between human and cynomolgus monkey 3A enzymes suggests similar enzyme behavior. However, no detailed kinetic and inhibition analyses of c3A4 have been performed thus far, although publications have detailed the expression and purification of c3A4 (Uno et al., 2007; Iwasaki et al., 2010; Ohtsuka et al., 2010; Emoto et al., 2011). In this manuscript, we present a novel human embryonic kidney (HEK) 293-6E system for the expression of c3A4 together with a detailed in vitro characterization of the enzyme. Species similarities and differences between h3A4 and c3A4 are also discussed in terms of substrate enzyme kinetics and inhibitory potencies.

Cloning of c3A4 and Cynomolgus NADPH-P450 Reductase.

c3A4 and cynomolgus CPR were cloned from cynomolgus monkey liver total RNA obtained from Biochain (Newark, CA). cDNA was prepared using Protoscript First Strand cDNA Synthesis Kit from New England BioLabs (Ipswich, MA). Primers for amplifying c3A4 were designed against the common chimpanzee (Pan troglodytes) CYP3A4 sequence, and primers for amplifying cynomolgus CPR were designed against the rhesus (Macaca mulatta) CPR sequence. KOD (Thermococcus kodakaraensis) enzyme (Novagen, Billerica, MA) was used for polymerase chain reaction (PCR) amplification, and PCR products were gel purified using Qiagen’s gel extraction kit (Venlo, The Netherlands). DNA fragments were cloned into a pTZ57R/T vector using standard molecular biology techniques, and clones were confirmed by DNA sequencing.

Mammalian Expression Constructs.

Both c3A4 and cynomolgus CPR were PCR amplified using their de novo clones as templates and cloned into a pDONR221 vector from Invitrogen to create an entry clone. Subsequently, these genes were moved into a pTT Gate vector which was a derivative of the original pTT vector (Durocher et al., 2002) through LR reaction using LR clonase from Invitrogen, as per the manufacturer’s protocol. Final expression constructs were sequence confirmed and used for transient transfections.

Expression of c3A4 in HEK293-6E Cells.

The HEK293-6E cells were cultured in serum-free F17 medium supplemented with 4 mM glutamine and 0.1% pluronic F68. The cells were maintained at 37°C in a 5% CO2 atmosphere with shaking at 130 rpm in the tissue culture incubator. On the day of transfection, the cells were seeded at a cell density of 1.1 × 106 cells/ml and cotransfected with 1 mg of c3A4 construct and 200 μg of P450 reductase construct (5:1 ratio) per liter of culture using PEI as a transfection reagent (Durocher et al., 2002). After 24 hours of transfection, cells were fed with 0.5% of tryptone N1 to increase recombinant protein production (Pham et al., 2005) and incubated for 48 hours.

Preparation of Microsomes from HEK293-6E Cells.

The transfected HEK293-6E cells were centrifuged at 3000 rpm for 15 minutes, and the pellet was washed with 1 × phosphate-buffered saline for 10 minutes. The cell pellet was resuspended in hypotonic buffer (100 mM potassium phosphate buffer, pH 7.5, 1 mM EGTA, 25 mM KCl, 10% glycerol), incubated for 20 minutes at 4°C, and spun at 1000 × g for 20 minutes. The cell pellet was further lysed using a dounce homogenizer in isotonic buffer containing 0.25 M sucrose and subjected to differential centrifugation at 1000 × g, 12,000 × g, and 100,000 × g. The final pellet was resuspended in 100 mM potassium phosphate, pH 7.5, and 10% glycerol and dialyzed against the same buffer overnight. All of the previously described steps were carried out at 4°C. The protein content was determined by Bradford’s method, and the sample was further analyzed by Western blot analysis and activity analyses.

Immunoblot Analysis.

Different concentrations of the microsome samples were electrophoresed by SDS-PAGE and transferred onto the nitrocellulose membrane. The blot was blocked with 5% skimmed milk powder for 2 hours at room temperature and then incubated with goat anti-h3A4 IgG (1:500) overnight. The blot was then incubated with donkey antigoat IgG-AP (1:5000) for 45 minutes at room temperature, and the bands were visualized using the nitro-blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate substrate. For detection of CPR, incubation with rabbit anti-CPR antibody (1:500) for 4 hours was followed by goat antirabbit IgG-AP (1:3000) for 1 hour.

Activity Determination.

Initial activity of the preparation was assessed by the formation of 1′-OH-MDZ upon incubation of the pellet with 20 µM MDZ in the presence of 2 mM NADPH in potassium phosphate buffer (pH 7.4) at 37°C for 30 minutes. Only pellets that showed robust activity and a discernible CO difference spectrum were considered for further scale-up. The CO difference spectrum was determined by performing a 20× to 100× dilution of the pellet in potassium phosphate buffer (100 mM, pH 7.4) and measuring the absorbance in a single-beam Tecan (Maennedorf, Switzerland) UV absorbance detector by scanning from 400 to 500 nm in 2-nm step sizes (Omura and Sato, 1964). An extinction coefficient of 11.99 was used for the determination of the 3A4 activity.

CPR Activity.

The amount of CPR present was determined as described previously (Venkatakrishnan et al., 2000). Briefly, 90 µl of 0.45 mg/ml cytochrome c, as a substrate, was mixed with 5 µl of differing concentrations of purified human CPR (standards) or 5 µl of c3A4 (unknown). Five microliters of NADPH (0.85 mg/ml) was added to catalyze this reaction, and the absorbance was measured at 550 nm continuously at 25°C. Absorbances of samples were subtracted from blanks not containing NADPH. The concentration of CPR in the samples was determined against a calibration curve of the purified human CPR (Sigma).

Analytical Methods.

A Waters Acquity UPLC (Ultra-performance Liquid Chromatography) system (Milford, MA) coupled to an API 4000 liquid chromatography–tandem mass spectrometry (AB-SCIEX, Toronto, Canada) was used for all bioanalyses. The mass spectrometry conditions are summarized in Supplemental Table 1. All analyses were conducted in positive mode ESI (Electrospray ionization). Standard curves for the metabolites were made by serial dilution in buffer. Separation was achieved on an Acquity BEH C18 (1.7 µM, 2.1*50 mm) UPLC column with mobile phase A consisting of 0.1% formic acid in water and mobile phase B consisting of 0.1% formic acid in acetonitrile. A flow rate of 0.6 ml/min was maintained starting at 85% A, and reduced to 50% A over 1 minute. From 1 to 1.5 minutes, the %A was reduced from 50% to 0%, and subsequently increased to 10% A from 1.5 to 1.7 minutes in a linear fashion. Mobile phase A (10%) was maintained for 0.1 minute and then increased to 85% A over 0.1 minute and kept at 85% A for another 0.1 minute for a total run time of 2 minutes. Alprazolam was used as the internal standard for all analyses.

Enzyme Kinetics.

Enzyme kinetics for MDZ, TST, terfenadine, nifedipine, and TRZ were performed by initially determining linear conditions of time and protein concentration in c3A4, h3A4, and MkLM for each substrate. The consumption of substrate was ensured to be less than 20%. MDZ and terfenadine kinetics were assessed with a concentration range of 0.015 to 100 µM in c3A4/h3A4 and up to 125 µM in MkLM for MDZ. The concentration range for TST, nifedipine, and TRZ was 0.78–200 µM in all systems. Standard incubation protocols were adopted wherein the appropriate amounts of protein and phosphate buffer (100 mM, pH 7.4) were mixed in a Waters 96-well deep plate, followed by the addition of substrate. This mix was preincubated at 37°C for 5 minutes before the addition of an appropriate volume of NADPH to give a final NADPH concentration of 2 mM. The protein concentration and incubation times are summarized with the results of the kinetics experiments in Table 1. The organic concentration in all incubations was less than 0.5%, and all experiments were performed in triplicates.

Kinetic characterization of multiple substrates after incubation with c3A4 and MkLM

The ability of c3A4 to metabolize non-h3A4 substrates was also assessed by performing a single-concentration incubation at 25 µM substrate, 50 nM c3A4, and 2 mM NADPH at 37°C for 30 minutes using the incubation protocol described earlier. The substrates and metabolites were monitored, and the results are summarized in Supplemental Table 2. 4′-OH-Diclofenac, dextrorphan, 1′-OH-bufuralol, acetaminophen, 7-hydroxycoumarin, hydroxybupropion, 6α -hydroxytaxol, and 6-OH-chlorzoxazone were the metabolites monitored when diclofenac, dextromethorphan, bufuralol, phenacetin, coumarin, bupropion, paclitaxel, and chlorzoxazone, respectively, were incubated with c3A4 to monitor the activity of 2C9, 2D6, 2D6, 1A2, 2A6, 2B6, 2C8, and 2E1, respectively. Expanded kinetics was performed for bufuralol, which was the only non-h3A4 substrate to show metabolism. This experiment was performed in both c3A4 and h3A4 with a 100 nM protein concentration and a 15-minute incubation time. The formation of 1′-OH-bufuralol was monitored as a surrogate for activity. The concentration range of bufuralol was 1–100 µM. To reconfirm the lack of metabolism of dextromethorphan, chlorzoxazone, and coumarin, the incubation was repeated at three concentrations of 50, 100, and 200 µM substrate in c3A4, MkLM, and h3A4 with a protein concentration of 50 nM for c3A4 and h3A4 and 1 mg/ml for MkLM. Other incubation variables remained identical to previously described conditions.

Inhibition Studies.

Inhibition of c3A4, h3A4, and MkLM was assessed using MDZ as a substrate at a concentration of 5µM, which was the determined substrate concentration at half of Vmax (Km) for MDZ. The protein concentration was 10 nM for c3A4/h3A4 and 0.5 mg/ml for MkLM with an incubation time of 10 minutes, conditions under which the reaction was determined to be in the linear range. The concentrations of KTZ ranged from 0.0025 to 0.16 µM with an extra solvent control not containing KTZ. The inhibition potency of TAO, a mechanism-based inhibitor, was determined with and without a primary incubation to determine the reversible and irreversible IC50, and the shifted IC50 value. The reversible IC50 experiment, performed in the absence of a primary incubation, was conducted with a protein concentration of 10 nM, MDZ concentration of 5 µM, and TAO concentrations ranging from 0.1 to 40 µM, with a 10-minute incubation. The irreversible IC50 was determined after a 20-minute primary incubation wherein 0.1–40 µM TAO was incubated with 10 nM c3A4/h3A4 in the presence of 2 mM NADPH. The secondary incubation was performed for 10 minutes with a further supplementation of NADPH and the addition of 5 µM MDZ (final concentration). The formation of 1′-OH-MDZ was a surrogate for activity remaining.

The inhibition of non-h3A4 inhibitors on MDZ activity was also assessed in c3A4 and MkLM. The list of inhibitors along with their concentration is summarized in Supplemental Table 3. Furafylline (20 µM), tranylcypromine (10 µM), clopidogrel (10 µM), montelukast (10 µM), sulphaphenazole (10 µM), benzylnirvanol (10 µM), quinidine (10 µM), and diethyldithiocarbamate (20 µM) were used to inhibit CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 2E1, respectively. The incubation contained 5 µM MDZ, 10 nM c3A4 or 1 mg/ml MkLM, and 2 mM NADPH and was performed for 30 minutes at 37°C in 100 mM KPO4 at pH 7.4. A 20-minute preincubation, in the presence of NADPH, was performed for furafylline since it is a known mechanism-based inhibitor of CYP1A2. The inhibitor concentrations were chosen to be many folds over their IC50 for h3A4.

Data Analyses.

The velocity versus substrate concentration data from the kinetics experiments were observed visually and from an Eadie-Hofstee plot, and subsequently fit to the appropriate equation. For typical hyperbolic kinetics, the Michaelis-Menten equation was used (eq. 1):

(1)

For biphasic kinetics, wherein a low Km and Vmax site and a high Km and Vmax site metabolize substrates simultaneously, eq. 2 was used (Tracy, 2003):

(2)

For substrate inhibition kinetics, wherein a low Vmax and high Km site predominates metabolism at high concentrations, eq. 3 was used (Tracy, 2003):

For a combination of allosteric and substrate inhibition, eq. 5 was derived and used:

(5)

In all these equations, v represents velocity of formation of a metabolite (amount of metabolite per unit time and unit protein concentration), Vmax the maximal rate of formation of a metabolite (enzyme capacity), S the substrate concentration, Km the substrate concentration at half of Vmax, Clint2 the slope of the linear portion of the biphasic graph (Clint2 = Vmax2/Km2 since saturation is not achievable), Ki the inhibition constant for substrate inhibition, and n Hill’s coefficient for sigmoidal kinetics. Subscripts of 1 or 2 indicate the first or second binding site, respectively. All fitting and analyses were performed using GraphPad Prism (version 5.02; GraphPad, La Jolla, CA).

IC50 values of inhibitors were determined by fitting eq. 6, a three-parameter inhibition model, in GraphPad Prism. The formation of 1′-OH-MDZ was monitored as a measure of activity remaining. An inhibitor-free solvent control was also run to ensure that greater than 80% inhibition was observed.

(6)

Results

Expression of Active c3A4.

Initially, c3A4 expression was attempted in Sf9 and Hi5 baculoviral cells, with multiple P450:CPR ratios, hemin concentrations, and addition times, but batch to batch variability in activities was observed. Use of a dual vector, with both c3A4 and cynomolgus CPR on the same construct, was also attempted, but the variability persisted. c3A4 was, subsequently, coexpressed with cynomolgus monkey CPR in HEK293-6E cells following the suggested protocol (Durocher et al., 2002), and this cell line afforded robust and reproducible activities. The HEK293 cell line, stably expressing Epstein-Barr virus nuclear antigen-1, increased the yield of both recombinant intracellular and secreted proteins. The use of cationic polymer PEI as transfection reagent helped in lowering the overall cost of the large-scale transient expression in HEK cells, whereas the addition of tryptone N1 (Organotechnie) increased the transient expression efficiency (Pham et al., 2005). After 48 hours of transfection, cells were harvested and microsomes were prepared using standard procedure involving hypotonic shock treatment. Upon expression and purification of c3A4, Western blots of the protein showed a band for c3A4 at around 55 kD and a band for reductase at around 72 kD, very similar to their respective molecular weights of 57 and 78 kD, respectively (Supplemental Fig. 1). The CO difference spectra showed an active c3A4 concentration of 1.4 µM (Supplemental Fig. 2) with a protein concentration of 13.5 mg/ml. A significant 420 nm peak was also observed, indicative of inactive protein. The concentration of reductase, as measured by the cytochrome c assay, was 1.0 µM. Hence, the ratio of c3A4 to cynomolgus CPR was approximate 3:2.

Enzyme Kinetics.

Full kinetic profiles were obtained for five substrates—MDZ, TST, terfenadine, nifedipine, and TRZ—after incubation with c3A4, h3A4, and MkLM.

MDZ with c3A4 showed pronounced atypical kinetics, with both homotrophic cooperativity (sigmoidal kinetics) and substrate inhibition being displayed (Fig. 1A). Data were fitted to the model described by eq. 5. The Vmax value obtained was 128 pmol/min/pmol-c3A4, the Km′ was 77 µM, and the Hill coefficient was 2.4 (Ki > 100 µM). Although a true Km could not be obtained since nonhyperbolic kinetics was observed, it was still possible to fit the data to a Michaelis-Menten equation (eq. 1) and obtain a composite Km of 6.3 µM. The Km and Vmax for MkLM were 1.5 µM and 61.6 pmol/min/mg protein, while the corresponding Km values for h3A4 and HLM are 1.6 and 4.2 µM. The Km values between all systems are hence comparable, and the low Km values suggest that MDZ is a high affinity substrate for both c3A4 and h3A4. The c3A4 Vmax was ∼9-fold higher than h3A4, confirming that this recombinant expressed enzyme is very active.

TST 6β-hydroxylation, another marker reaction for h3A4, was also efficient in c3A4 and MkLM (Fig. 1B). Km values of 49.2 and 67.2 µM were obtained in c3A4 and MkLM, respectively, whereas the corresponding Km values for h3A4 and HLM are 78 and 46.4 µM (Walsky and Obach, 2004). While the reaction was hyperbolic in the presence of c3A4, sigmoidal kinetics was observed with MkLM (eq. 4). The reaction appeared to be faster in c3A4 than h3A4, with a Vmax of 446 compared with 86 pmol/min/pmol-P450. However, the relative ratios of reductase and b5 can impact the velocity of the reaction. In contrast, the reaction was faster in HLM as compared with MkLM (5260 versus 3481 pmol/min/mg), although this is on a per milligram basis. The Km values are similar across all four systems, making TST 6β-hydroxylation an attractive in vitro probe substrate to compare activities between humans and cynomolgus monkeys.

Terfenadine alcohol formation showed hyperbolic kinetics (eq. 1) in c3A4 and sigmoidal kinetics (eq. 4) in MkLM (Fig. 1C). Terfenadine was an efficient c3A4 substrate, with a very low Km of 0.5 µM and Vmax of 31 pmol/min/pmol-c3A4. The corresponding Km and Vmax values in MkLM were 7.2 µM and 116 pmol/min/mg-MkLM, respectively (Table 1). Similar kinetics have been reported previously in the literature, with Km values ranging from 3 to 12 mM for h3A4 and HLM (Rodrigues et al., 1995). The Km value for c3A4 was substantially lower than the three other comparative systems, while the Vmax of c3A4 was similar to h3A4. The low Km in c3A4 as compared with MkLM may suggest the involvement of a second lower affinity enzyme in terfenadine oxidation, or altered nonspecific protein binding of the terfenadine to the milieu in MkLM studies.

c3A4 efficiently catalyzed the oxidation of nifedipine to oxidized nifedipine with a low Km of 2.8 µM, which was around 10-fold lower than the MkLM Km (Fig. 1D; Table 1), while MkLM, h3A4, and HLM had similar Km values. Substrate inhibition was observed in c3A4 (eq. 4), whereas sigmoidal plus substrate inhibition was observed in MkLM (eq. 5), similar to the mixed-atypical kinetics seen when MDZ was incubated with c3A4. The Vmax values were comparable between c3A4 and h3A4, but HLM had a 5-fold higher Vmax than MkLM (Patki et al., 2003). The low Km in c3A4 as compared with MkLM may suggest the involvement of a second enzyme/altered nonspecific protein binding, as in the case of terfenadine.

TRZ 1- and 4-hydroxylation kinetics were hyperbolic (eq. 1) in all cases except in MkLM, where 1-hydroxylation showed biphasic kinetics (eq. 2). TRZ 1-hydroxylation is shown in Fig. 1E, whereas TRZ 4-hydroxylation is shown in Fig. 1F. TRZ 1-hydroxylation had a lower Km and lower Vmax than 4-hydroxylation in HLM and h3A4 (Patki et al., 2003). The same trend was observed in MkLM as well, but in c3A4, 1- and 4-hydroxylations had similar Vmax and Km values.

To determine whether c3A4 can metabolize non-h3A4 substrates, a single concentration of substrate was incubated as described in the Materials and Methods section. Of all the probe substrates for CYP2C9, 2D6, 1A2, 2A6, 2B6, 2C8, and 2E1 tested, only bufuralol (CYP2D6 substrate) showed any metabolism (Supplemental Table 2). Hence, a follow-up multisubstrate concentration experiment was performed with bufuralol concentrations ranging from 1 to 100 µM with both c3A4 and h3A4 (Fig. 2). Saturation was not achieved even at the highest bufuralol concentration in c3A4, suggesting a very high Km reaction. The maximal velocity of 1′-OH-bufuralol formation was 0.4 pmol/min/pmol-c3A4, suggestive of a reasonable amount of metabolism. h3A4 showed no metabolism of bufuralol across all concentration ranges.

Enzyme kinetics of bufuralol when incubated with c3A4 and h3A4. 1′-OH-bufuralol velocities are on the y-axis, whereas the x-axis represents bufuralol concentration.

Table 1 summarizes the time and protein conditions for each Vmax and Km determination, the Vmax and Km values, and the corresponding data in HLM and h3A4 from the literature.

Enzyme Inhibition.

Inhibition of c3A4 by KTZ (reversible) and TAO (mechanism-based) was also performed. The KTZ IC50 of 1′-OH-MDZ formation was 0.03 µM in c3A4 and 0.016 µM in MkLM (Table 2). The corresponding values for h3A4 and HLM are 0.04 and 0.02, respectively (Walsky and Obach, 2004). Hence, all the IC50 values are similar to each other, confirming the suitability of KTZ as a c3A4 inhibitor at low inhibitor concentrations.

TAO also behaved as a time-dependent inhibitor in c3A4, similar to h3A4, MkLM, and HLM. The c3A4 IC50 at 0 minute was 1.2 µM, suggesting competitive inhibition, and the IC50 at 30 minutes was 0.17 µM, an 8-fold IC50 shift suggesting time-dependent inhibition (Fig. 3; Table 2). The corresponding IC50 shift in MkLM was 9-fold, although the IC50 numbers at time zero and time 30 minutes were ∼15-fold higher (Fig. 3; Table 2). The effect of non-h3A4 inhibitors on c3A4 was also determined using the selective inhibitors for the various human CYPs (Supplemental Table 3). As can be seen from the table, none of the non-h3A4 inhibitors inhibited MDZ 1′-hydroxylation in c3A4 and MkLM even at concentrations several fold over their IC50 values for their specific P450. This suggests that non-h3A4 inhibitors are unlikely to inhibit c3A4.

Inhibition of c3A4 after a 0-minute (A) and 20-minute (B) preincubation with TAO, and inhibition of MkLM after a 0-minute (C) and 20-minute (D) preincubation with TAO. The x-axis contains the log concentrations of TAO, whereas the y-axis contains 1′-OH-MDZ concentrations, as a marker for c3A4 activity remaining.

Discussion

c3A4 has been previously expressed in Escherichia coli, and some characterization has been completed with several substrates at one or more concentrations (Uno et al., 2007; Iwasaki et al., 2010; Ohtsuka et al., 2010; Emoto et al., 2011). However, extensive kinetic characterization with multiple substrates, multiconcentration characterization with non-h3A4 substrates, and inhibition propensity with non-h3A4 inhibitors has not been previously described. This paper attempts to provide such data and, as a result, provide a broad data set to understand c3A4 and its similarities and differences compared with h3A4. In addition, this is the first report of HEK293 cells being used to express c3A4, a robust system that may prove useful to researchers attempting to express P450s of different species that are not amenable to expression in baculoviral cells.

Several reports have provided c3A4 activity information for h3A4 and non-h3A4 substrates at one or two substrate concentrations (Iwasaki et al., 2010; Emoto et al., 2011). For example, the alprazolam 4-hydroxylation kinetic profile was characterized for c3A4 and h3A4, and was shown to display sigmoidal kinetics (Ohtsuka et al., 2010) in both cases. c3A4 was characterized by an ∼8-fold higher Vmax, a ∼2-fold lower Km, and a corresponding 13-fold higher intrinsic clearance over h3A4.

Extensive characterization of rhesus monkey CYP3A64 (proposed renaming to CYP3A4), expressed in a baculoviral system, was accomplished using TST, nifedipine, MDZ, and benzoxy-4-trifluoromethylcoumarin as substrates, and it was found that Vmax, Km, and Clint values were very similar between rhesus 3A64 and h3A4 for these four substrates (Carr et al., 2006). Sigmoidal kinetics were also observed for some of these substrates, and KTZ was found to inhibit 3A64 metabolism with an IC50 identical to h3A4 (TST as substrate). Given the 100% homology between c3A4 and rhesus 3A64 enzymes, it is unsurprising that the Km values obtained were similar for the common substrates between our study and the rhesus 3A64 study (Carr et al., 2006). Collectively, these data suggest that rhesus 3A64, h3A4, and c3A4 are characterized by similar kinetic properties.

From our studies, for MDZ, low Km values (<10 µM) were obtained in c3A4, MkLM, h3A4, and HLM, suggesting that this is a high affinity substrate across species. A combination of sigmoidal kinetics at the low substrate concentrations and substrate inhibition at the high substrate concentrations was observed in c3A4, and hence the profile was fitted to an equation derived from combining the sigmoidal and substrate inhibition equations. A similar profile was also noted for nifedipine in MkLM. The existence of atypical kinetics suggests the existence of multiple catalytic sites on c3A4 as with h3A4. With TST as a substrate, the Km values for c3A4, h3A4, HLM, and MkLM were similar to each other, whereas with nifedipine and terfenadine as substrates, c3A4 had a Km value approximately 10-fold lower than h3A4, HLM, and MkLM. For the latter two substrates, HLM, MkLM, and h3A4 had similar Km values, suggesting that c3A4 has higher affinity or lower nonspecific protein binding for these two substrates. With TRZ as a substrate, c3A4, h3A4, and MkLM were equally efficient in catalyzing both 1′ and 4′-hydroxylation pathways, whereas in MkLM, 4′-hydroxylation was about 3-fold more efficient that 1′-hydroxylation in terms of Vmax/Km values.

When the microsomal Vmax values are converted from a per milligram to a per picomole basis using the relative abundance ratios of P450s, direct comparisons can be made between expressed enzyme Vmax values and microsomal Vmax values (Rodrigues, 1999; Uehara et al., 2011). These values are summarized in Table 3. The ratio for expressed enzyme Vmax to microsome Vmax was consistent between humans and cynomolgus monkeys for three out of four substrates (TST, terfenadine, and nifedipine). Viewed this way, c3A4 and h3A4 behaved very similarly for three substrates. Indeed, MDZ is extensively used as an in vivo probe substrate for CYP3A4 activity in both humans and cynomolgus monkeys and shows similar hepatic extraction ratios in both species (Akabane et al., 2010; Yoda et al., 2012).

While c3A4 shows an almost exclusive preference to metabolize h3A4 substrates, earlier reports have indicated that c3A4 and c3A5 could metabolize dextromethorphan and bufuralol (2D6 substrates), in addition to chlorzoxazone (2E1 substrate). Surprisingly, h3A4 was also able to catalyze these reactions (Iwasaki et al., 2010; Emoto et al., 2011). At 20 µM bufuralol concentration, h3A4 and c3A4 appeared to be equally proficient at catalyzing 1′-hydroxylation, whereas c3A5 was 3–4 times more efficient than h3A5 in catalyzing the reaction. This trend was more pronounced at 200 µM bufuralol. MkLM showed 8-fold higher bufuralol 1′-hydrxoylation activity than HLM at 20 and 200 µM bufuralol concentrations. Bufuralol 6-hydroxylation occurred with equal velocities in liver microsomes at both concentrations in both species (Iwasaki et al., 2010; Emoto et al., 2011). Hence, bufuralol 6-hydroxylation does not show a species difference, and bufuralol 1′-hydroxylation species difference appears to be more due to c3A5 than c3A4. Dextromethorphan O-dealkylation was catalyzed only by c3A4 and c3A5 and not by h3A4/3A5, and MkLM also showed higher velocities than HLM (Emoto et al., 2011). Dextromethorphan N-demethylation reaction velocity was similar in MkLM and HLM, and both species 3A4s were able to catalyze this reaction (Emoto et al., 2011). Hence, only the dextromethorphan O-dealkylation was a clear species difference, although the higher activity (85- to 400-fold) of the 2D enzymes over 3A enzymes may ameliorate the species difference. Chlorzoxazone was only tested at 50 µM, and at this concentration, c3A4/5 showed 4- to 5-fold higher velocities than h3A4/5, although HLM and MkLM showed similar velocities (Iwasaki et al., 2010; Emoto et al., 2011). It is unclear if this is truly a species difference, since 50 µM is a very high concentration.

In our studies, 25 µM dextromethorphan did not show the formation of dextrorphan, and 25 µM chlorzoxazone did not show the formation of 1-OH-chlorzoxazone. These findings were reconfirmed in a repeat experiment conducted in triplicates at higher substrate concentrations of 50–200 µM. In contrast, bufuralol showed the formation of 1′-OH-bufuralol when incubated with c3A4. While bufuralol showed minor metabolism (<1%) when incubated at 25 µM, in a repeat multiconcentration study, a maximal velocity of 0.4 pmol/min/pmol-c3A4 was suggestive of low-level metabolism. Saturation of metabolism was not observed, and h3A4 showed no metabolism of bufuralol across all concentration ranges. Consistently in microsomes, MkLM Vmax values for bufuralol 1′-OH hydroxylation was found to be 15-fold higher than HLM Vmax values, suggesting higher intrinsic bufuralol clearance in MkLM as compared with HLM (Mankowski et al., 1999). The c2D17 and c2D44 Vmax for bufuralol hydroxylation ranges between 10 and 20 pmol/min/pmol-2D6 (Uno et al., 2010). The maximal velocity is 0.4 pmol/min/pmol-c3A4 by c3A4, but the abundance of c3A4 (39 pmol/mg) is more than 10-fold higher than c2D (3.3 pmol/mg); hence, the amount adjusted to c3A4 (15.6 pmol/min/mg protein) rate is much closer to the cynomolgus CYP2D6 bufuralol hydroxylation rate (33–66 pmol/min/mg protein), and hence, c3A4 may play a significant role in bufuralol hydroxylation in MkLM.

In summary, we have extensively characterized a preparation of c3A4 and compared it to both h3A4 and MkLM. Enzyme kinetic parameters were obtained for five prototypical h3A4 substrates, and in all cases, c3A4 showed robust activity with some differences in Km values. In addition, the inhibition parameters for c3A4 with two prototypical CYP3A inhibitors (KTZ and TAO) were comparable to those obtained with h3A4. c3A4 was also able to metabolize bufuralol to its 1-hydroxylated metabolite, although no metabolism of dextromethorphan or chlorzoxazone was observed.